Universal system, method and solution for the acceleration of the process of fixing, dehydrating and clearing the structure of biological tissue

ABSTRACT

Described herein are methods, compositions, kits and systems for fixing biological samples. In one aspect, the method includes contacting the tissue sample with a fixing composition comprising about 20 to about 80% v/v DMSO. The solutions of the dehydration and clearing steps also include DMSO. Tissues fixed according to the methods described herein (and using the compositions herein described) may be fixed at a more rapid rate as compared to conventional procedures, but with comparable results.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/557,758 filed Dec. 2, 2014, which claims priority to U.S. ProvisionalPatent Application No. 61/938,189, filed Feb. 11, 2014. This applicationis incorporated herein by reference in its entirety.

BACKGROUND

For microscopic analysis, a few cell types are thin enough to be vieweddirectly (e.g., algae, protozoa, blood, tissue cultures), but mosttissues (e.g., kidney, liver, brain) are too thick to allow light to betransmitted through them. Thus, in order to be examined with amicroscope, a specimen must be sufficiently thin to be transparent andmust possess sufficient contrast to permit the resolution of structuraldetail. The tissues can be sliced into very thin sections provided theyare first processed to prevent cell damage.

Commonly, two types of procedures are used in preparing specimens oftissue for microscopic examination. In one procedure a specimen isfrozen, cut and mounted on a slide in an elapsed time of about 15minutes. This so-called “frozen section” procedure has the advantage ofenabling a rapid histological diagnosis to be made from the specimen,and it is frequently employed in situations where a diagnosis isnecessary while a patient is on an operating table. However, the “frozensection” procedure possesses certain disadvantages in that the preparedslide does not possess the uniformity of quality of slides prepared byother methods. Thus, frozen sections are difficult to interpret, and aremore likely to be misinterpreted by a pathologist than are usualpermanent slides. Further, the process of freezing tissue introducesconsiderable artifacts in it that can make certain conditions, such assome cancers, impossible to diagnose. Thus, when the frozen sectionprocedure is used in emergency situations, it is customary for anotherportion of the tissue specimen to be processed using standardfixation/paraffin embedding techniques to have tissue available foradditional sections if further examination becomes necessary.

In the other procedures, a slide of relatively high quality is producedwhen a section of the specimen is chemically fixed and mounted in ablock of paraffin. However, using conventional procedures and solutions,the time required to process a specimen of tissue for mounting inparaffin is on the order of many hours to days as compared with theminutes required to process a specimen by the frozen section procedure.

In the preparation of paraffin slides, a specimen of tissue is immersedinitially in a fixing agent. The fixed specimen is then immersed in adehydrating agent, and afterward the specimen is immersed in a clearingagent. Finally, the cleared specimen is immersed in a bath of paraffinwhich impregnates the specimen and permits it to be sliced into thinsections for subsequent mounting onto slides. Because of the length oftime required to prepare specimens by this process, it is customary forhospital laboratories to begin processing the specimens late in theafternoon after surgeons have obtained specimens from their patients.The processing continues through the night, and slides of the specimensare available for microscopic examination the next morning. Although theslides produced according to this procedure are of higher quality thanthose produced by the frozen section technique, the length of timerequired to process specimens is too great to enable this procedure tobe used in situations where time is of the essence.

Using current procedures, if time or enzyme function is critical frozensections are the preferred process. If subcellular detail is important,other procedures must be used. Selection of the correct proceduredepends on what the cell biologist is looking for and to a point,becomes an art form.

What is needed in the art is a method of fixing tissue which combinesthe time advantage of the frozen section with the consistency andquality of the traditional paraffin embedded tissue.

SUMMARY OF THE INVENTION

In one aspect, a method of fixing a biological tissue sample isprovided. The method includes contacting the tissue sample with a fixingcomposition comprising about 20-80% v/v DMSO for a time ranging from 10seconds to 24 hours. The method further includes dehydrating the tissuesample; and clearing the tissue sample. In one embodiment, the sample iscontacted with the fixing composition for 30 seconds to 5 minutes. Inone embodiment the fixed tissue is then embedded in paraffin,nitrocellulose, plastic or other suitable embedding agent.

In another aspect, a method of fixing a biological tissue sample isprovided. The method includes contacting the tissue sample with adehydration solution composition comprising about 2-80% v/v DMSO for atime ranging from 10 seconds to 24 hours. In one embodiment, the sampleis contacted with a series of dehydration solutions containingincreasingly higher percentages of alcohol. In one embodiment, thesample is contacted with a solution containing about 50% alcohol andabout 50% DMSO. In another embodiment, the sample is then contacted witha solution containing about 98% alcohol and about 2% DMSO. Additionalsteps may be included between the two previously identified steps. Theconcentrations discussed below with regard to dehydration solutions areappropriate for use in this embodiment. The method further includesclearing the tissue sample. In one embodiment, the sample is contactedwith the dehydration composition for 30 seconds to 5 minutes. In oneembodiment, the fixed tissue is contacted with a clearing agent. Inanother embodiment the fixed tissue is then embedded in paraffin,nitrocellulose, plastic or other suitable embedding agent.

In a further aspect, a method for fixing a tissue sample is provided.The method includes (a) contacting the tissue sample with a fixingcomposition for about 1 minute to about 5 minutes, the fixingcomposition comprising about 40% v/v DMSO; about 10% v/v of formalin;and a buffering agent; (b) dehydrating the tissue sample in anon-aqueous solution comprising DMSO and alcohol; and (c) clearing thetissue sample in a clearing solution comprising xylene and DMSO. In oneembodiment, the method includes (d) embedding the tissue in paraffin,nitrocellulose, plastic or other suitable material.

In another aspect, a kit for use in preparing tissues is provided. Inone embodiment, the kit includes one or more of: a fixative solution, adehydration solution and a clearing solution. In one embodiment, thefixative solution includes 20-80% DMSO, a fixing agent and a bufferingagent. In another embodiment, the dehydration solution includes one ormore alcohols and DMSO. In a further embodiment, the clearing solutionincludes xylene and DMSO. In another embodiment, the kit includes morethan one fixative solution, more than one dehydration solution, and/ormore than one clearing solution. In a further embodiment, not all of thefixative solution, dehydration solution and clearing solution is presentin the kit.

In another aspect, a system for tissue fixing is provided. In oneembodiment, the system includes any of the components of the kitsdescribed herein, in conjunction with one or more mechanical aids, asfurther described herein.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a control human colon section prepared according to the methoddescribed in Example 3.

FIG. 2 is a different control human colon section prepared according tothe method described in Example 3.

FIG. 3 is a human colon section prepared according to the methoddescribed in Example 2.

FIG. 4 is the human colon section of FIG. 3, magnified 20×.

FIG. 5 is a human lung section prepared according to the methoddescribed in Example 2.

FIG. 6 is the human lung section of FIG. 5, magnified 20×.

FIG. 7 is a human fatty liver section prepared according to the methoddescribed in Example 2.

FIG. 8 is the human fatty liver section of FIG. 7, magnified 20×.

FIG. 9 is a human spleen section prepared according to the described inExample 2.

FIG. 10 is the human spleen liver section of FIG. 9, magnified 20×.

FIG. 11 is a human heart section prepared according to the methoddescribed in Example 2.

FIG. 12 is the human heart section of FIG. 11, magnified 20×.

FIG. 13 is a control human colon section prepared according to themethod described in Example 3.

FIG. 14 is the control human colon section of FIG. 13, magnified 20×.

FIG. 15 is a control human colon section prepared according to themethod described in Example 3.

FIG. 16 is the control human colon section of FIG. 15, magnified 20×.

FIG. 17 is a control human colon section prepared according to a lessoptimal fixation/processing procedure of the prior art.

FIG. 18 is the control human colon section of FIG. 17, magnified 20×.

FIG. 19 is a control human colon section prepared according to a lessoptimal fixation/processing procedure of the prior art.

FIG. 20 is the control human colon section of FIG. 19, magnified 20×.

FIG. 21 is a mouse smooth muscle section prepared according to themethod described in Example 2.

FIG. 22 is the mouse smooth muscle section of FIG. 21, magnified 20×.

FIG. 23 is a mouse liver section prepared according to the method asdescribed in Example 2.

FIG. 24 is the mouse liver section of FIG. 21, magnified 20×.

FIG. 25 is a mouse heart section prepared according to the method asdescribed in Example 2.

FIG. 26 is the mouse heart section of FIG. 25, magnified 20×.

FIG. 27 is a mouse skeletal muscle section prepared according to themethod described in Example 2.

FIG. 28 is the mouse skeletal muscle section of FIG. 27, magnified 20×.

FIG. 29 is a mouse heart section prepared according to the methoddescribed in Example 2.

FIG. 30 is the mouse heart section of FIG. 29, magnified 20×.

FIG. 31 is a mouse lung section prepared according to the methoddescribed in Example 2.

FIG. 32 is the mouse lung section of FIG. 31, magnified 20×.

FIG. 33 is a mouse kidney section prepared according to the methoddescribed in Example 2.

FIG. 34 is the mouse kidney section of FIG. 33, magnified 20×.

FIG. 35 is a human colon section prepared according to the methoddescribed in Example 2, stained for cytokeratin.

FIG. 36 is a control human colon section prepared according to themethod described in Example 3, stained for cytokeratin.

FIG. 37 is a human lymphoma section prepared according to the methoddescribed in Example 2, stained for human leukocyte antigen.

DETAILED DESCRIPTION OF THE INVENTION

It is to be noted that the term “a” or “an” refers to one or more. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein.

While various embodiments in the specification are presented using“comprising” language, under other circumstances, a related embodimentis also intended to be interpreted and described using “consisting of”or “consisting essentially of” language. The words “comprise”,“comprises”, and “comprising” are to be interpreted inclusively ratherthan exclusively. The words “consist”, “consisting”, and its variants,are to be interpreted exclusively, rather than inclusively.

As used herein, the term “about” means a variability of 10% from thereference given, unless otherwise specified. Each of the rangesspecified herein are inclusive of the endpoints. For example, a range of20% to 40% includes 20%, 40% and all of the values in between.

As used herein, “disease”, “disorder” and “condition” are usedinterchangeably, to indicate an abnormal state in a subject.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art and by reference to published texts, whichprovide one skilled in the art with a general guide to many of the termsused in the present application.

The methods, compositions, kits and systems described herein, providethe ability to produce high quality stained tissue sections at afraction of the time required for conventional procedures. During cancersurgery, the ability to provide a pathological diagnosis from a stainedtissue section will provide the surgeon with information that may beused prior to the patient's departure from the operating room (i.e.,intraoperative diagnosis). For example, an indication from the anatomicpathologist that the cancer is confined to the resected tissue may allowthe surgeon to be conservative in treatment and to preserve neighboringhealthy tissue. Alternatively, a finding by the anatomic pathologistthat cancer is not confined to a resected organ would permit moreaggressive surgical treatment while a patient is still in the operatingroom.

Formalin fixation and paraffin embedding are conventional tissuepreservation and processing methods used for histologic diagnosis inover 90% of cases. However, formalin fixation is a slow procedure(approximately 1 h per 1 mm of tissue penetration). Overall, thecompositions, methods, systems and kits described herein can providerapid and improved morphological and molecular preservation to betteraccommodate both traditional and molecular diagnoses.

Tissue Sample

In one embodiment, a method for fixing and stabilizing a cell's nativemorphology, in a cell containing sample, is provided. The methods,compositions and kits described herein are suitable for use in fixingany desired biological sample which may require stabilization. As usedherein, the term “biological sample” may be used interchangeably with“tissue”. While, in one embodiment, the methods, compositions, and kitsdescribed herein are useful with tissues, such as tissue from a biopsy,it is to be understood that any biological sample which containscells—whether or not normally classified as a “tissue”—may be fixedusing the methods described herein.

In one embodiment, the biological tissue is any tissue that may be fixedusing conventional techniques. In another embodiment, the tissue sampleis animal or plant tissue. An animal tissue may be derived from a humanor non-human animal, including, without limitation, non-human primates,dogs, cats, mice, rats, guinea pigs, fruit flies, and other laboratoryanimals; farm animals including bovine, ovine, horse, goat, pig, rabbit,etc. In one embodiment, the tissue is from a mammal. In anotherembodiment, the tissue is from a human. Tissues may be derived from bothlive and dead sources, e.g., from an autopsy. In another embodiment, thetissue is from an organ, biopsy, circulating tumor cell (CTC), bloodsample, plasma sample, serum sample, tissue culture cells, saliva,urine, cerebral spinal fluid, medical sample, egg, embryo, or adulttissue. Tissue samples may be fresh or previously frozen, e.g., thoseobtained from tissue banks or previously held in storage. Solid tissuemay be obtained from surgical biopsy or resection.

Exemplary tissues that may be processed include: appendix, bladder,bone, bowel, brain, breast, carcinoma, cervix (squamous epithelium),gall bladder, heart, kidney, liver, lung, ovary, parotid gland,placenta, prostate, skin, spleen, testicle, thyroid gland, tonsil, anduterus (myometrium and endometrium). Lymphoreticular and fatty tissuesmay also be processed. Optionally, mineralized tissue may requiredecalcification prior to processing by the present method. Subsequentanalysis may include, without limitation, detecting DNA mutations andRNA expression, genomic analysis, histochemistry, immunochemistry, andproteomic analysis.

Any size tissue sample may be processed using the methods, compositionsand kits described herein. Processing times may vary based, in part, onthe size of the sample. In one embodiment, the tissue sample is about0.1 to about 2.0 cm in size, or fractions or integers therebetween. Inanother embodiment, the tissue sample is about 1 to about 5 mm in size,or fractions or integers therebetween. In another embodiment, the tissuesample is about 1 mm. In another embodiment, the tissue sample is about2 mm. In another embodiment, the tissue sample is about 3 mm. In anotherembodiment, the tissue sample is about 4 mm. In another embodiment, thetissue sample is about 5 mm. In another embodiment, the tissue sample isabout 6 mm. In another embodiment, the tissue sample is about 7 mm. Inanother embodiment, the tissue sample is about 8 mm. In anotherembodiment, the tissue sample is about 9 mm. In another embodiment, thetissue sample is about 10 mm.

Fixing

In one aspect, a method of fixing biological tissue is provided. In oneembodiment, a fixing solution is use which contains from about 20% toabout 80% dimethyl sulfoxide (DMSO). The methods and kits providedherein are based on, in part, that relatively high concentrations ofDMSO, when included in a fixing solution, allow the fixative topenetrate much more rapidly than without DMSO. This providessignificantly faster processing times for fixing biological tissues.Further, the inclusion of DMSO helps increase the strength and speed ofcrosslinking of proteins, when used with an additive fixing agent, andprovide clear, consistent specimens for imaging.

In one embodiment, the fixing solution includes about 20% to about 80%DMSO, inclusive of the endpoints. In yet another embodiment, the fixingsolution includes about 30% to about 60% DMSO. In another embodiment,the fixing solution includes about 40% to about 50% DMSO. In yet anotherembodiment, the fixing solution includes about 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, or about 80% DMSO. The DMSO used maybe of commercial grade, e.g., about 95% or greater purity. In oneembodiment, the DMSO is of about 99% or greater purity.

In another embodiment, the fixing solution further includes one or morefixing agents. In one embodiment, the fixing solution includes twofixing agents. In one embodiment, the fixing solution includes threefixing agents. In one embodiment, the fixing solution includes fourfixing agents. In one embodiment, the fixing solution includes five ormore fixing agents. The fixing agent may be any chemical fixative, manyof which are known in the art. See, e.g., Kiernan, Histological andHistochemical Methods, 4^(th) Ed., Scion Publishing 2008, which isincorporated herein by reference. In one embodiment, the fixing agent isformaldehyde. In another embodiment, the fixing agent includes formalin.A saturated water solution, of about 40% formaldehyde by volume or 37%by mass, is called “100% formalin”. A small amount of stabilizer, suchas methanol, may be added to suppress oxidation and polymerization. Atypical commercial grade formalin may contain about 10-12% methanol inaddition to various metallic impurities. A commercial grade formalinsolution may also be buffered. In one embodiment, the formalin solutioncontains sodium phosphate, monobasic and/or sodium phosphate, dibasic.

Common fixative agents which are useful in the invention include,without limitation, formaldehyde, formalin, paraformaldehyde,gluteraldehyde, other aldehydes including chloral hydrate, acrolein,hydroxyaldipaldehyde, crotonaldehyde and glyoxal; organic coagulantsincluding alcohols including ethanol and methanol; acetone;tricholoroacetic acid; mercuric chloride; zinc salts including zincchloride; mercuric chloride; picric acid; acetic acid; chromiumcompounds including chromium trioxide and potassium dichromate; osmiumtetroxide; and ruthenium tetroxide. Other fixative agents useful in theinvention include mineral acids that serve as coagulants; metal ions andcomplexes that cause precipitation of proteins; iodine; organic proteincoagulants including ρ-toluenesulphonic acid and tannic acid;surfactants; cationic dyes; bifunctional organic compounds; and sodiumperiodate. Other non-formalin based commercially available fixativeagents include FineFIX, RCL2, and HOPE-in.

The selection of the fixing agent may be determined by the person ofskill in the art based on the desired structural or chemical componentsand the end use of the fixed tissue sample. Often a mixture of differentfixing agents is employed in order to offset undesirable effects ofindividual substances and to obtain more than one type of chemicalfixation. In one embodiment, acetic acid may be included in the fixativesolution to preserve chromosomes, to precipitate chromatin of interphasenuclei, and to oppose the shrinking actions of other agents such asethanol and picric acid.

The concentration of the fixing agent will be determined based on theselection of the agent and the end use of the fixed tissue sample, andranges from about 0.25% (for gluteraldehyde) to up to about 80% (foralcohols). In one embodiment, the fixative is formaldehyde. In anotherembodiment, the formaldehyde includes about 2% to 10% of the fixingsolution. In one embodiment, the fixative includes about 5 to about 20%formalin. In one embodiment, the fixative includes about 10% formalin.In one embodiment, the fixative includes about 5% formalin. In oneembodiment, the fixative includes about 15% formalin. In one embodiment,the fixative includes about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20% formalin. In another embodiment, the fixative is analcohol. In one embodiment, the fixative is ethanol, methanol, orisopropanol, or combinations thereof. In one embodiment, the fixative isa combination of ethanol, methanol, and isopropanol. In anotherembodiment, the alcohol or mixture thereof includes about 30% to about80% of the fixing solution. In another embodiment, the fixative isacetone. In another embodiment, the acetone includes about 30% to about80% of the fixing solution. In another embodiment, the fixative isgluteraldehyde. In another embodiment, the gluteraldehyde includes about0.25% to about 4% of the fixing solution. In another embodiment, thefixative is glyoxal. The concentration of the fixing agent can bedetermined by the person of skill in the art.

In one embodiment, the fixing solution includes more than one fixingagent. Specific combinations of fixing agents are known in the art andmay be selected by the person of skill in the art. Some combinationsknown in the art, which are useful in the invention, include withoutlimitation: ethanol and glacial acetic acid; ethanol, chloroform, andglacial acetic acid; ethanol, formalin, and glacial acetic acid;formalin and sodium chloride; neutral buffered formalin; formaldehydeand gluteraldehyde; formalin and calcium acetate; formalin and zincsulfate; picric acid, formalin and glacial acetic acid; picric acid,formalin, ethanol, ethyl acetate, and glacial acetic acid; mercuricchloride, sodium chloride, trichloroacetic acid, formalin and glacialacetic acid; mercuric chloride, potassium dichromate, and sodiumsulfate, optionally with formalin and/or glacial acetic acid; osmiumtetroxide and potassium dichromate, optionally with glacial acetic acid;osmium tetroxide and mercuric chloride; paraformaldehyde and picricacid; lysine monohydrochloride, sodium phosphate, paraformaldehyde andsodium metaperiodate or sodium paraperiodate; calcium acetate, zincacetate and zinc chloride. Other combinations of the above-identifiedfixatives and others are also contemplated for use in the invention.

The fixative solution may also include one or more additionalcomponents. In one embodiment, the fixative solution includes one ormore buffering agents. Suitable buffers include those that maintain thepH of the fixing solution in the range of about 4 to about 9. In oneembodiment, the fixing solution is maintained at a physiological pH. Inone embodiment, a pH in the range of about 7 to about 8 is obtained. Inanother embodiment, the fixing solution is about pH 7.4. The bufferingagent may be organic or non-organic, based upon the composition of theremainder of the fixing composition. Fixing compositions comprisingabout 40 to about 45% or greater DMSO may require an organic bufferingagent, although this can be readily determined by one of skill in theart. Organic buffering agents known in the art and useful in theinvention include, without limitation, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MES(2-(N-morpholino)ethanesulfonic acid), and TRIS, also known as THAM(tris(hydroxymethyl)aminomethane)). Non-organic buffering agents knownin the art and useful in the invention include, without limitation,potassium hydrogen phthalate, borax, boric acid, sodium acetate, HCl,acetic acid, disodium hydrogen phosphate, sodium dihydrogen phosphate,potassium dihydrogen phosphate, citric acid, sodium citrate, sodiumcacodylate, barbitone sodium, blycine, sodium hydroxide, sodiumcarbonate, and sodium bicarbonate.

In one embodiment, the fixing solution may include one or more of thecomponents listed as clearing agents, as further described below. In oneembodiment, the fixing solution includes xylene or a xylene substitute.In another embodiment, the fixing solution includes naphtha.

The fixing solution may further contain one or more salts includingsodium chloride, potassium chloride, calcium chloride, magnesiumchloride, and the like. In addition, in one embodiment, the fixingsolution includes one or more solvent. The solvent may be water, saline,phosphate buffered saline, Ringer's solution, etc., or combinationsthereof.

In one embodiment, the fixing solution includes about 4% to about 10%formaldehyde, about 40% to about 50% DMSO, water, and a buffering agent.In one embodiment, the buffering agent includes one or more of sodiumphosphate, monobasic and sodium phosphate, dibasic. In anotherembodiment, the buffer includes tetrapropyl ammonium phosphate.

In the method of fixing described herein, the tissue may be fixed for atime ranging from about 10 seconds to about 24 hours. The duration ofthe fixing step will be determined based on the size of the tissuesample, the fixing solution used, the temperature and whether or not anymechanical aids are used. In another embodiment, the fixing step isabout 20 seconds. In another embodiment, the fixing step is about 30seconds. In another embodiment, the fixing step is about 40 seconds. Inanother embodiment, the fixing step is about 50 seconds. In anotherembodiment, the fixing step is about 1 minute. In another embodiment,the fixing step is about 75 seconds. In another embodiment, the fixingstep is about 90 seconds. In another embodiment, the fixing step isabout 105 seconds. In another embodiment, the fixing step is about 2minutes. In another embodiment, the fixing step is about 2 minutes and30 seconds. In another embodiment, the fixing step is about 2 minutesand 45 seconds. In another embodiment, the fixing step is about 3minutes. In another embodiment, the fixing step is about 3 minutes and15 seconds. In another embodiment, the fixing step is about 3 minutesand 30 seconds. In another embodiment, the fixing step is about 3minutes and 45 seconds. In another embodiment, the fixing step is about4 minutes. In another embodiment, the fixing step is about 4 minutes and30 seconds. In another embodiment, the fixing step is about 5 minutes.In another embodiment, the fixing step is about 5 minutes and 30seconds. In another embodiment, the fixing step is about 6 minutes. Inanother embodiment, the fixing step is about 6 minutes and 30 seconds.In another embodiment, the fixing step is about 7 minutes. In anotherembodiment, the fixing step is about 7 minutes and 30 seconds. Inanother embodiment, the fixing step is about 8 minutes. In anotherembodiment, the fixing step is about 8 minutes and 30 seconds. Inanother embodiment, the fixing step is about 9 minutes. In anotherembodiment, the fixing step is about 9 minutes and 30 seconds. Inanother embodiment, the fixing step is about 10 minutes. In yet anotherembodiment, the fixing step is 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20minutes. In yet another embodiment, the fixing step is 20, 25, 30, 35,40, 45, 50, 55 or 60 minutes. In yet another embodiment, the fixing stepis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours.

In one embodiment, the “fixing step” as described above, is performedusing an alcohol as the fixing agent. Thus, it can be said that, in thatembodiment, the fixing step is skipped and dehydration is the first stepof the method. In this embodiment, it is appropriate that any of thedehydration solutions/steps described herein may be used as the “fixing”step/agent. In one embodiment, the dehydration includes about 2% toabout 60% DMSO.

Decalcification

In one embodiment, the tissue is decalcified after fixing. The tissuemay be decalcified by acid or chelating agent. For acid decalcificationthe choice of acid may be selected from those known in the art. Knowndecalcifying acids include, without limitation, formic acid, De Castro'sfluid (nitric acid), ascorbic acid, acetic acid. For decalcification bychelation, EDTA is used. In one embodiment, disodium EDTA is used. Inanother embodiment, ammonium EDTA is used.

Hard specimens cannot be sectioned with an ordinary microtome, but theycan be softened after fixation, usually by removing the substancesresponsible for the hardness of the tissue. Decalcification is thechemical dissolution of insoluble calcium salts with a suitable acid orchelating agent. Other approaches are available for softening hardmaterials that contain silica or which owe their hardness to compactorganic materials. Alternatively, hard tissues can be cut with a specialmicrotome, which is equipped with a chisel-shaped knife of hardenedsteel.

Dehydration

In one embodiment, the tissue is dehydrated after fixing, afterdecalcification, or both. A biological tissue specimen may be embeddedin paraffin once it has been equilibrated with a solvent that ismiscible with wax. This is typically accomplished by replacing the waterin the specimen, first with alcohol and then with a paraffin solvent(clearing agent).

In one embodiment of the methods described herein, the dehydrationsolution includes DMSO. DMSO may be present in the dehydration solutionfrom an amount of about 1% to about 60%. In one embodiment, thedehydration solution includes about 2% to about 30% DMSO. In anotherembodiment, the dehydration solution includes about 5% DMSO. In anotherembodiment, the dehydration solution includes about 10% DMSO. In anotherembodiment, the dehydration solution includes about 15% DMSO. In anotherembodiment, the dehydration solution includes about 20% DMSO. In anotherembodiment, the dehydration solution includes about 25% DMSO. In anotherembodiment, the dehydration solution includes about 30% DMSO. In anotherembodiment, the dehydration solution includes about 35% DMSO. In anotherembodiment, the dehydration solution includes about 40% DMSO. In anotherembodiment, the dehydration solution includes about 45% DMSO. In anotherembodiment, the dehydration solution includes about 50% DMSO. In anotherembodiment, the dehydration solution includes about 55% DMSO. In anotherembodiment, the dehydration solution includes about 60% DMSO. In yetanother embodiment, the dehydration solution includes about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30% DMSO. In yet another embodiment, thedehydration solution includes about 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60% DMSO.

In one embodiment, the dehydration step is performed in a non-aqueoussolution comprising DMSO. The non-aqueous solution may include more thanone component. In one embodiment, the non-aqueous solution includes oneor more alcohols. The alcohol, in one embodiment, is ethanol,isopropanol, methanol, or combinations thereof. In another embodiment,the non-aqueous solution includes acetone. In another embodiment, thenon-aqueous solution includes one or more alcohol.

In one embodiment of the invention, the dehydration step includesmultiple steps, each successive step comprising contacting the samplewith a higher percentage of alcohol. For example, in one embodiment, thedehydration solution of includes three separate solutions: (i) about 70%alcohol and about 30% DMSO; (ii) about 95% alcohol and about 5% DMSO;and (iii) about 98% alcohol and about 2% DMSO. In one embodiment, thesample is contacted with the first dehydration solution for a period oftime; then the sample is contacted with the second dehydration solutionfor a second period of time; then the sample is contacted with the thirddehydration solution for a third period of time.

In the methods described herein, the tissue may be dehydrated for a timeranging from about 10 seconds to about 24 hours. The duration of thedehydration step(s) will be determined based on the size of the tissuesample, the dehydration solution used, the temperature and whether ornot any mechanical aids are used. The duration of each dehydration step,if applicable, may be individually selected. In one embodiment, thedehydration step is about 20 seconds. In another embodiment, thedehydration step is about 30 seconds. In another embodiment, thedehydration step is about 40 seconds. In another embodiment, thedehydration step is about 50 seconds. In another embodiment, thedehydration step is about 1 minute. In another embodiment, thedehydration step is about 75 seconds. In another embodiment, thedehydration step is about 90 seconds. In another embodiment, thedehydration step is about 105 seconds. In another embodiment, thedehydration step is about 2 minutes. In another embodiment, thedehydration step is about 2 minutes and 30 seconds. In anotherembodiment, the dehydration step is about 2 minutes and 45 seconds. Inanother embodiment, the dehydration step is about 3 minutes. In anotherembodiment, the dehydration step is about 3 minutes and 15 seconds. Inanother embodiment, the dehydration step is about 3 minutes and 30seconds. In another embodiment, the dehydration step is about 3 minutesand 45 seconds. In another embodiment, the dehydration step is about 4minutes. In another embodiment, the dehydration step is about 4 minutesand 30 seconds. In another embodiment, the dehydration step is about 5minutes. In another embodiment, the dehydration step is about 5 minutesand 30 seconds. In another embodiment, the dehydration step is about 6minutes. In another embodiment, the dehydration step is about 6 minutesand 30 seconds. In another embodiment, the dehydration step is about 7minutes. In another embodiment, the dehydration step is about 7 minutesand 30 seconds. In another embodiment, the dehydration step is about 8minutes. In another embodiment, the dehydration step is about 8 minutesand 30 seconds. In another embodiment, the dehydration step is about 9minutes. In another embodiment, the dehydration step is about 9 minutesand 30 seconds. In another embodiment, the dehydration step is about 10minutes. In yet another embodiment, the dehydration step is 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 minutes. In yet another embodiment, thedehydration step is 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes. In yetanother embodiment, the dehydration step is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours. Ifthe dehydration step includes more than one step, each step may be equalor different in duration.

Clearing

In one embodiment, the fixed tissue is contacted with a clearingsolution which includes a solvent which is miscible with the embeddingagent. Xylene is useful in the methods described herein and is misciblewith paraffin. Xylene (or xylenes) is the common name for the chemicaldimethylbenzene. In one embodiment, xylene may be a mixture of the threeisomers 1,2-dimethylbenzene (o-xylene), 1,3-dimethylbenzene (m-xylene)and 1,4-dimethylbenzene (p-xylene). Laboratory-grade xylene may containm-xylene (about 40 to about 65%), p-xylene (about 20%), o-xylene (about20%) and ethyl benzene (about 6 to 20%) and, optionally, traces oftoluene, trimethyl benzene, phenol, thiophene, pyridine and hydrogensulfide. Xylene containing other ratios of the three isomer forms isalso useful herein. Reagent-grade xylene is also useful herein. Otherclearing agents useful herein include, without limitation, benzene,choloroform, toluene and mixtures thereof. Other clearing agents include‘xylene substitutes’, including D-, L- and DL-limonene. Other clearingagents include amyl acetate, n-butanol, carbon tetrachloride, cedarwoodoil, white oil, n-heptane, benzyl benzoate, methyl benzoate, methylsiacylate, terpineol, methyl chloroform and mixtures thereof. Additionalclearing agents include t-butanol, dioxane, dimethyl sulfoxinde, THF andmixtures thereof. Further clearing agents include proprietary solutionsincluding HistoChoice® clearing agent, XS-3™, ClearRite™, Clearify™,etc. The clearing agent may include combinations of any of the listedagents.

In one embodiment, the clearing solution includes DMSO. DMSO may bepresent in the clearing solution from an amount of about 2% to about20%. In one embodiment, the clearing solution includes about 10% DMSO.In one embodiment, the clearing solution includes about 2% to DMSO. Inanother embodiment, the clearing solution includes about 3% DMSO. Inanother embodiment, the clearing solution includes about 4% DMSO. Inanother embodiment, the clearing solution includes about 5% DMSO. In oneembodiment, the clearing solution includes about 6% to DMSO. In anotherembodiment, the clearing solution includes about 7% DMSO. In anotherembodiment, the clearing solution includes about 8% DMSO. In anotherembodiment, the clearing solution includes about 9% DMSO. In oneembodiment, the clearing solution includes about 11% to DMSO. In anotherembodiment, the clearing solution includes about 12% DMSO. In anotherembodiment, the clearing solution includes about 13% DMSO. In anotherembodiment, the clearing solution includes about 14% DMSO. In oneembodiment, the clearing solution includes about 15% to DMSO. In anotherembodiment, the clearing solution includes about 16% DMSO. In anotherembodiment, the clearing solution includes about 17% DMSO. In anotherembodiment, the clearing solution includes about 18% DMSO. In oneembodiment, the clearing solution includes about 19% to DMSO. In anotherembodiment, the clearing solution includes about 20% DMSO.

In one embodiment, the clearing step includes multiple steps. In eachstep, the composition of the clearing solution may be individuallydetermined. In one embodiment, the clearing step includes contacting thesample with a solution comprising xylene and DMSO.

In one embodiment, the dehydration and clearing steps are combined. Inanother embodiment, the dehydration and clearing steps are alternated.For example, in one embodiment, the clearing step includes contactingthe sample with the following solutions: (i) about 98% xylene and about2% DMSO; (ii) about 95% alcohol and about 5% DMSO; (iii) about 98%xylene and about 2% DMSO.

In one embodiment, the method for fixing a tissue sample includes:

-   -   (a) contacting the tissue sample with a fixing composition for        about 1 minute to about 5 minutes, the fixing composition        comprising about 40% v/v DMSO; about 10% v/v of formalin; and a        buffering agent;    -   (b) dehydrating the tissue sample in a non-aqueous solution        comprising DMSO and alcohol;    -   (c) clearing the tissue sample in a clearing solution comprising        xylene and DMSO; and    -   (d) infiltrating with and embedding the tissue in paraffin,        nitrocellulose, plastic or other suitable material.

Temperature

When the temperature of a fixative is raised or lowered, the rate ofdiffusion into the specimen is affected, as is the rate of the chemicalfixation reactions occurring with the various tissue components.Increasing temperature accelerates the process of fixation. Excessiveheat however, particularly if it is prolonged, can damage cells andcause substantial shrinkage and hardening of the specimen. Another issuewith using hot fixative solutions to initially fix larger specimens(greater than 3 mm thick), is that the outside of the specimen fixesrapidly whilst it may take quite some time for the fixative to penetrateto the center of the block and this area may be poorly fixed or notfixed at all. Thus, the temperature at which the methods describedherein are performed, may be selected by the person of skill in the artbased on the desired speed, size of the specimen, etc.

The methods described herein may be performed at any temperature inwhich the reagents (in combination) remain in liquid form. For example,although the freezing point of DMSO is relatively high at about 18.5°C., when in combination with other reagents, the solutions are useful attemperatures below this point. In one embodiment, any of the stepsdescribed herein are performed at a temperature from 0° C. to about 80°C. The temperature at which each step is performed may be selectedindividually for that step. In one embodiment, the method is performedat room temperature In another embodiment, the steps are performed atabout 37° C. to about 45° C. In another embodiment, the steps areperformed at about 40 to about 55° C. In yet another embodiment, thesteps are performed at about 46° C.

Mechanical Aids

Various mechanical aids are known in the art which may aid in theoverall fixation process, including any of the steps herein described.Mechanical aids useful herein include, without limitation, microwave,ultrasound, vacuum, agitation, electrophoresis, centrifuge, andautomated tissue processors. Microwaves are a form of non-ionizingradiation produced by the magnetron in domestic and scientific microwaveovens. At a frequency of 2.5 GHz, they have the capacity to generateinstantaneous heat when dipolar molecules such as water or polar sidechains of proteins are exposed to their alternating magnetic fields at2.5 billion cycles per second. The rate at which the microwave energywill generate heat during tissue fixation depends on a number of factorsincluding the power setting and power output of the oven, the volume andnature of the holding solution, the composition, shape and number ofcontainers (including cassettes), the agitation or movement of thecontainers, and the number, volume and dimensions of the specimens beingfixed. Thus, the use of microwaves will result in shorter fixationtimes. In conjunction with the method and compositions described herein,the use of microwave technology can further speed the fixation process.

In one embodiment, a microwave is used in any of the methods describedherein. For example, tissue specimens in a DMSO containing fixativesolution described herein may be microwaved to assist the fixativeaction of the fixing agent (referred to as “microwave-assistedfixation”). In one embodiment, the microwaving may be carried out whilethe tissue is in fixative, in which case there may be some hazard fromtoxic fumes produced, or the tissue may be transferred to saline orbuffer for the microwave step. In this embodiment, the use of fixativesof relatively low toxicity containing glyoxal, are useful in the methodsdescribed herein. In one embodiment, the tissue sample is contacted witha fixing composition containing about 20 to about 80% v/v DMSO, asdescribed herein. The tissue sample is then microwaved for about 5seconds to about 5 minutes. In one embodiment, the sample is microwavedin the fixative. In another embodiment, the sample is transferred to abuffer or saline solution prior to microwaving. In one embodiment, thetissue sample is microwaved for about 10 seconds. In another embodiment,the tissue sample is microwaved for about 15 seconds. In anotherembodiment, the tissue sample is microwaved for about 20 seconds. Inanother embodiment, the tissue sample is microwaved for about 25seconds. In another embodiment, the tissue sample is microwaved forabout 30 seconds. In another embodiment, the tissue sample is microwavedfor about 40 seconds. In another embodiment, the tissue sample ismicrowaved for about 50 seconds. In another embodiment, the tissuesample is microwaved for about 60 seconds. In another embodiment, thetissue sample is microwaved for about 75 seconds. In another embodiment,the tissue sample is microwaved for about 90 seconds. In anotherembodiment, the tissue sample is microwaved for about 2 minutes. Inanother embodiment, the tissue sample is microwaved for about 2.5minutes. In another embodiment, the tissue sample is microwaved forabout 3 minutes. In another embodiment, the tissue sample is microwavedfor about 3.5 minutes. In another embodiment, the tissue sample ismicrowaved for about 4 minutes. In another embodiment, the tissue sampleis microwaved for about 4.5 minutes.

Ultrasound technology may also be employed in conjunction with any ofthe methods described herein. The critical factors for consistentultrasound-facilitated processing without tissue damage are maintainingultrasound at a high frequency (>0.1 MHz) and high intensity (1-20W/cm2) and controlling the total energy received by the tissue See,e.g., Wang et al, Ultrasound Facilitated Formalin-fixed andParaffin-embedded Tissue Specimen Preparation Technology, 2008 BRNSymposium Poster available atbiospecimens.cancer.gov/meeting/brnsymposium/docs/2008pres/Poster27-Wang.pdf,which is incorporated by reference herein. Applying high-frequency,high-intensity ultrasound to the fixative cuts fixation time. Theintensity of ultrasound used depends on the particular procedure andwill be in the range of about 0.001-20 W/cm2. See, e.g., U.S. Pat. No.6,291,180, which is incorporated by reference herein. Fixation ofvarious tissues such as lymph node, brain, breast, and prostate suggeststhat, compared to the conventional method, implementation of ultrasoundretains superior and more uniform tissue morphology preservation. Lessprotein antigenicity is altered so that rapid immunohistochemicalreactions occur with higher sensitivity and intensity, reducing the needfor antigen retrieval pretreatment. Better RNA preservation results instronger signals in in situ hybridization and longer RNA fragmentsextracted from fixed tissues, probably due to rapid inhibition ofendogenous RNase activity. See, Chu et al, Ultrasound-acceleratedformalin fixation of tissue improves morphology, antigen and mRNApreservation, Modern Pathology (2005) 18, 850-863, advance onlinepublication, 17 Dec. 2004, which is incorporated by reference herein.The use of ultrasound in conjunction with the methods described hereincan be readily accomplished by one of skill in the art. In oneembodiment, ultrasound is used in conjunction with microwave technologyin the methods described herein.

In one embodiment, the tissue sample is contacted with a fixingcomposition containing about 20 to about 80% v/v DMSO, as describedherein. The tissue sample is then subject to high intensity, highfrequency ultrasound for about 5 seconds to about 15 minutes. In oneembodiment, the sample is subject to ultrasound while in the fixative.In another embodiment, the sample is transferred to a buffer or salinesolution prior to ultrasound. In one embodiment, the tissue sample issubject to ultrasound for about 10 seconds. In another embodiment, thetissue sample is subject to ultrasound for about 15 seconds. In anotherembodiment, the tissue sample is subject to ultrasound for about 20seconds. In another embodiment, the tissue sample is subject toultrasound for about 25 seconds. In another embodiment, the tissuesample is subject to ultrasound for about 30 seconds. In anotherembodiment, the tissue sample is subject to ultrasound for about 40seconds. In another embodiment, the tissue sample is subject toultrasound for about 50 seconds. In another embodiment, the tissuesample is subject to ultrasound for about 60 seconds. In anotherembodiment, the tissue sample is subject to ultrasound for about 75seconds. In another embodiment, the tissue sample is subject toultrasound for about 90 seconds. In another embodiment, the tissuesample is subject to ultrasound for about 2 minutes. In anotherembodiment, the tissue sample is subject to ultrasound for about 2.5minutes. In another embodiment, the tissue sample is subject toultrasound for about 3 minutes. In another embodiment, the tissue sampleis subject to ultrasound for about 3.5 minutes. In another embodiment,the tissue sample is subject to ultrasound for about 4 minutes. Inanother embodiment, the tissue sample is subject to ultrasound for about4.5 minutes. In another embodiment, the tissue sample is subject toultrasound for about 5 minutes. In another embodiment, the tissue sampleis subject to ultrasound for about 6 minutes. In another embodiment, thetissue sample is subject to ultrasound for about 7 minutes. In anotherembodiment, the tissue sample is subject to ultrasound for about 8minutes. In another embodiment, the tissue sample is subject toultrasound for about 9 minutes. In another embodiment, the tissue sampleis subject to ultrasound for about 10 minutes. In another embodiment,the tissue sample is subject to ultrasound for about 11 minutes. Inanother embodiment, the tissue sample is subject to ultrasound for about12 minutes. In another embodiment, the tissue sample is subject toultrasound for about 13 minutes. In another embodiment, the tissuesample is subject to ultrasound for about 14 minutes. In anotherembodiment, the tissue sample is subject to ultrasound for about 15minutes.

The methods described herein may also utilize an automated tissueprocessor. There are two main types of processors, the tissue-transfer(or “dip and dunk”) machines where specimens are transferred fromcontainer to container to be processed, or the fluid-transfer (or“enclosed”) types where specimens are held in a single process chamberor retort and fluids are pumped in and out as required. Most modernfluid-transfer processors employ raised temperatures, effective fluidcirculation and incorporate vacuum/pressure cycles to enhance processingand reduce processing times.

In one embodiment, an automated tissue processor is used with thefixative, dehydration, and/or clearing solutions described herein. Inanother embodiment, the methods described herein are carried out usingan automated tissue processor.

Various automated tissue processors which are useful with the methodsdescribed herein, are known in the art. Some exemplary proprietaryprocessors include TissueWave™ 2 Microwave Processor, Excelsior™ ASTissue Processor, STP 120 Spin Tissue Processor, all from ThermoScientific; Tissue Processing; Leica ASP6025, Leica PELORIS II, LeicaRemoteCare, Leica ASP300 S, Leica ASP200 S, and Leica TP1020, all fromLeica Biosystems; LYNX II from Electron Microscopy Sciences, etc. Othersystems are known in the art and are equally useful herein.

Kits

In another aspect of the invention, a kit for use in preparing specimensis provided. In one embodiment, the kit includes any of the componentsdescribed herein. In another embodiment, the kit contains one or more ofthe following components: (a) a fixative solution comprising about 20 toabout 80% DMSO, a fixing agent and a buffering agent; (b) a dehydrationsolution comprising one or more alcohols and DMSO; and (c) a clearingsolution comprising xylene and DMSO. In another embodiment, the kitincludes more than one of component (a) and/or more than one ofcomponent (b) and/or more than one of component (c). In one embodiment,the fixative agent is formaldehyde (formalin), gluteraldehyde, glyoxalor alcohol. In another embodiment, the kit includes more than onedehydration solution, each solution comprising a different percentage ofalcohol. In one embodiment, the fixative solution of (a) includes about40% DMSO.

In another embodiment, the kit provided herein includes (a) a fixativesolution comprising about 40% DMSO, a fixing agent and a bufferingagent; (b) a dehydration solution comprising one or more alcohol andDMSO; and (c) a clearing solution comprising xylene and DMSO. In oneembodiment, the dehydration solution of (b) includes multiple solutions,each solution comprising a different percentage of alcohol. In anotherembodiment, the dehydration solution of (b) includes: (i) about 70%alcohol and about 30% DMSO; (ii) about 95% alcohol and about 5% DMSO,and (iii) about 98% alcohol and about 2% DMSO

In another embodiment, the kit includes one or more components necessaryfor embedding the tissue in paraffin, nitrocellulose, plastic or othersuitable embedding agent.

In addition to the above components, the kit may include vessels forcollecting the specimen, instructions, various tubes, etc.

The methods, solutions, and kits described herein are suitable for usewith any commonly used or commercially available immunohistochemistrymethods/kits. The sections produced as described herein may be stainedaccording to standard protocols, using standard reagents. The sectionsproduced according to the invention are of equal or superior quality tosections produced according to commonly used fixation methods.

The following examples are illustrative only and are not intended tolimit the present invention.

EXAMPLES Example 1 Samples Fixed According to One Embodiment of theMethod of the Invention

The following biopsy tissue samples were obtained: human colon, humanlung, human fatty liver, human spleen, human heart, mouse smooth muscle,mouse liver, mouse heart, mouse skeletal muscle, mouse lung, mousekidney, human lymphoma Tissue samples approximately 2 mm in size wereprocessed according to the following protocol, at 46° C.:

1 minute in a fixing solution containing 10% v/v formalin (buffered withsodium phosphate, monobasic and/or sodium phosphate, dibasic), 40% DMSOin water. 1 minute in a dehydration solution containing 70% alcohol and30% DMSO. 1 minute in a dehydration solution containing 95% alcohol and5% DMSO. 1 minute in a dehydration solution containing 98% alcohol and2% DMSO. 1 minute in a clearing solution containing 90% xylene and 10%DMSO. 1 minute in a dehydration solution containing 98% alcohol and 2%DMSO. 1 minute in a clearing solution containing 90% xylene and 10%DMSO.

The samples were then infiltrated with paraffin for 15 minutes at about60° C. The samples were then embedded in paraffin according to standardprotocols. The samples were sectioned on a Manual Rotary Microtome forRoutine Sectioning (Thermo Scientific™ HM 325) to a thickness of 4 μM.Sections were de-paraffinized and stained using standardimmunohistochemical techniques (e.g., haematoxylin and eosin stain).

In brief, the de-paraffinizing and staining protocol were as follows:

1. xylene—6 min;

2. alcohol (ethanol) 100%—60 sec;

3. alcohol (ethanol) 95%—30 sec;

4. rinse with water—60 sec;

5. Hematoxylin—60 sec;

6. rinse with water—90 sec;

7. alcohol (ethanol) 95%—30 sec;

8. Eosin—30 sec;

9. alcohol (ethanol) 95%—10 sec;

10. alcohol (ethanol) 100%—90 sec;

11. Xylene—60 sec.

Samples were coverslipped and viewed under a microscope. Slides werefound to be comparable to slides prepared according to commonly usedprocedures (data not shown).

Example 2 Samples Fixed According to One Embodiment of the Method of theInvention

Tissue samples approximately 4 mm in size were fixed and processedaccording to the following protocol, at 46° C.:

2.5 minute in a fixing solution containing 10% v/v formalin (bufferedwith sodium phosphate, monobasic and/or sodium phosphate, dibasic), 40%DMSO in water. 2.5 minutes in a dehydration solution containing 70%alcohol and 30% DMSO. 2.5 minutes in a dehydration solution containing95% alcohol and 5% DMSO. 2.5 minutes in a dehydration solutioncontaining 98% alcohol and 2% DMSO. 2.5 minutes in a clearing solutioncontaining 90% xylene and 10% DMSO. 2.5 minutes in a dehydrationsolution containing 98% alcohol and 2% DMSO. 2.5 minutes in a clearingsolution containing 90% xylene and 10% DMSO.

The samples were then infiltrated with paraffin for 15 minutes at about60° C. The samples were then embedded in paraffin according to standardprotocols. The samples were sectioned on a Manual Rotary Microtome forRoutine Sectioning (Thermo Scientific™ HM 325) to a thickness of 4 μM.Sections were de-paraffinized as described in Example 2 and stainedusing standard immunohistochemical techniques (e.g., haematoxylin andeosin stain).

Samples were coverslipped and viewed under a microscope. Slides werefound to be comparable to slides prepared according to commonly usedprocedures. Slides showing the 4 μm sections prepared according to thismethod can be seen in FIGS. 5-12, 21-35 and 37.

Example 3 Control Slides

Briefly, all samples are biopsied and placed into containers containing10% formalin. Upon receipt by the lab (up to 18 hours later), thesamples were subject to the following procedure, which is carried out at37° C.

Formalin 10%—10 hours

Alcohol (ethanol), 70%—30 minutes

Alcohol (ethanol), 95%—45 minutes

Alcohol (ethanol), 95%—45 minutes

Alcohol (ethanol), 100%—1 hour

Alcohol (ethanol), 100%—1 hour

Alcohol (ethanol), 100%—1 hour

Xylene—1 hour

Xylene—1 hour

The sample was then infiltrated with paraffin (2 changes) for 45 mineach. This was performed at about 60° C. The samples were then embeddedin paraffin according to standard protocols. The samples were sectionedon a microtome to a thickness of 4 μM.

The section was then de-paraffinized according to standard procedures.In brief: the sample was immersed for 2 minutes in histology gradexylene. This was performed 4 times. Now that the paraffin is removed,the excess xylene is removed. The sample is immersed for 1 minute inabsolute ethanol (2×). The sample is immersed for 30 seconds in 95%ethanol. The sample is immersed for 45 seconds in 70% ethanol. Thesample is washed with water for 1 minute. The slides were stainedaccording to standard procedures.

Slides labeled “Control” were prepared using these standard proceduresand can be found in FIGS. 1-4, 13-20 and 36.

The methods, compositions, kits and systems described hereinsufficiently decrease the time of the process of fixing, dehydrating andclearing of the structure of biological tissue; sufficiently improve thequality of the fixed structure of the biological tissue; prevent anyartifacts; maintain the histologic structure on the biologic tissue; andimprove the quality of the image of the structure of the biologicaltissue.

All publications cited in this specification are incorporated herein byreference. While the invention has been described with reference toparticular embodiments, it will be appreciated that modifications can bemade without departing from the spirit of the invention. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A method of fixing a biological tissue sample,the method comprising: (a) contacting the tissue sample with a fixingcomposition, the fixing composition comprising about 40% v/v DMSO andabout 12% formalin; (b) dehydrating the tissue sample; and (c) clearingthe tissue sample.
 2. The method of claim 1, further comprising: (d)infiltrating the tissue with an embedding agent; and (e) embedding thetissue in the embedding agent.
 3. The method of claim 2, wherein theclearing step is performed in a clearing solution which comprises asolvent which is miscible with the embedding agent.
 4. The method ofclaim 1, wherein the dehydration step is performed in a non-aqueoussolution comprising DMSO.
 5. The method of claim 4, wherein thenon-aqueous solution comprises one or more alcohols.
 6. The method ofclaim 5, wherein the dehydration step comprises multiple steps, eachsuccessive step comprising contacting the sample with a higherpercentage of alcohol.
 7. The method of claim 1, wherein the clearingstep is performed in a clearing solution which comprises DMSO.
 8. Themethod of claim 1, wherein the clearing step is performed in a clearingsolution which comprises xylene.
 9. The method of claim 1, wherein theclearing step comprises multiple steps.
 10. The method of claim 1,wherein the fixing step is performed for about 10 seconds.
 11. Themethod of claim 1, wherein the fixing step is performed for about 30seconds.
 12. The method of claim 1, wherein the fixing compositionfurther comprises one or more of water, xylene, xylene substitute, ornaphtha.
 13. The method of claim 1, wherein the tissue sample of step(a) is frozen.
 14. The method of claim 1, wherein the tissue iscontacted with the fixing composition for about 10 seconds to about 24hours.
 15. A method of fixing a frozen biological tissue sample, themethod comprising: (a) contacting the tissue sample with a fixingcomposition, the fixing composition comprising about 40% v/v DMSO andabout 12% formalin; (b) dehydrating the tissue sample; and (c) clearingthe tissue sample.
 16. The method of claim 15, wherein the tissue iscontacted with the fixing composition for about 10 seconds to about 24hours.
 17. The method of claim 15, further comprising: (d) infiltratingthe tissue with an embedding agent; and (e) embedding the tissue in theembedding agent.
 18. The method of claim 15, wherein the dehydrationstep is performed in a non-aqueous solution comprising DMSO.
 19. Themethod of claim 15, wherein the clearing step is performed in a clearingsolution which comprises DMSO.
 20. The method of claim 15, wherein thefixing composition further comprises one or more of water, xylene,xylene substitute, or naphtha.